Phospholipases A2 (PLA2) are a superfamily of enzymes that catalyze the rate-limiting step in the production of eicosanoids that are potent mediators of inflammation. Because an imbalance in the production of these potent mediators of allergic and inflammatory reactions could lead to acute and chronic inflammatory diseases, including sepsis, rheumatoid arthritis and asthma, understanding the regulation of PLA2 is an important step toward the elucidation of the pathogenesis of inflammatory diseases. Among many forms of mammalian secretory PLA2s and intracellular PLA2s identified so far, secretory group V PLA2 (gVPLA2) and cytosolic group IVA PLA2 (cPLA2-a) have been implicated in eicosanoid biosynthesis and inflammation. On the basis of progress made during the last granting period in terms of technology development and mechanistic understanding, we propose to continue our research on these two enzymes to fully understand the mechanisms of their actions and regulation under different pathophysiological conditions, with an eventual goal of developing PLA2-specific anti-inflammatory agents. Specific aims during this proposed period are: (1) to determine the mechanisms of cellular entry and intracellular actions of gVPLA2, (2) to study the mechanisms of transcellular actions of gVPLA2, and finally (3) to determine the mechanisms by which multi-site phosphorylation regulates cPLA2-a activities. The principal methodologies to be used include: (1) surface plasmon resonance analysis that allows for real-time measurement of membrane-protein and protein-protein binding, (2) real-time cellular fluorescence assays for gVPLA2 and cPLA2-a, (3) real-time fluorescence monitoring of cellular membrane translocation of fluorescently labeled gVPLA2 and cPLA2-a, and (4) isolation and identification of specific binding proteins for cPLA2-a.